Various analytical procedures and devices are commonly employed in flow-through assays to determine the presence and/or concentration of analytes that may be present in a test sample. For instance, immunoassays utilize mechanisms of the immune systems, wherein antibodies are produced in response to the presence of antigens that are pathogenic or foreign to the organisms. These antibodies and antigens, i.e., immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism that can be used to determine the presence or concentration of that particular antigen in a biological sample.
There are several well-known immunoassay methods that use immunoreactants labeled with a detectable component so that the analyte can be detected analytically. For example, “sandwich-type” assays typically involve mixing the test sample with detectable probes, such as dyed latex or a radioisotope, which are conjugated with a specific binding member for the analyte. The conjugated probes form complexes with the analyte. These complexes then reach a zone of immobilized antibodies where binding occurs between the antibodies and the analyte to form ternary “sandwich complexes.” The sandwich complexes are localized at the zone for detection of the analyte. This technique can be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich-type assays are described in. by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al.
However, many conventional “sandwich-type” assay formats encounter significant inaccuracies when exposed to relatively high analyte concentrations. Specifically, when the analyte is present at high concentrations, a substantial portion of the analyte in the test sample may not form complexes with the conjugated probes. Thus, upon reaching the detection zone, the uncomplexed analyte competes with the complexed analyte for binding sites. Because the uncomplexed analyte is not labeled with a probe, it cannot be detected. Consequently, if a significant number of the binding sites become occupied by the uncomplexed analyte, the assay may exhibit a “false negative.” This problem is commonly referred to as the “hook effect.”
Various techniques for reducing the “hook effect” in immunoassays have been proposed. For instance, U.S. Pat. No. 6,184,042 to Neumann, et al. describes one technique for reducing the hook effect in a sandwich assay. The technique involves incubating the sample in the presence of a solid phase with at least two receptors capable of binding to the analyte. The first receptor is an oligomer of a binding molecule selected from antibodies, antibody fragments and mixtures thereof. The second receptor is bound to or capable of being bound to a solid phase. The use of a soluble oligomeric antibody is said to reduce the “hook effect.”
A need still exists, however, for an improved technique of reducing the “hook effect” in a simple, efficient, and relatively inexpensive manner.